The objective of this project is to define events associated with phorbol ester action, beginning with occupancy of the phorbol ester receptor in hormone-responsive cells. Particular emphasis is placed on the coupling of phorbol ester receptor occupancy with phorbol ester activation of protein kinase C. A model cell culture system has been used to study the process by which cells become refractory to phorbol ester-directed responses. The results indicate that the observed desensitization is not due to a biochemical change in the receptor/protein kinase C. Certain phospholipid mixtures were found to inhibit protein kinase C activity at limiting Ca++ concentrations without inhibiting binding, suggesting a role for phospholipid metabolism in regulation of coupling of phorbol ester binding and activation of protein kinase C. Several factors which interfere with either the binding or the kinase assay have been identified. Although no role for these in regulation of kinase or binding is known, characterization of these factors has facilitated quantitation of the kinase and binding activities in crude and partially purified preparations. The role of diacylglycerol in activation of protein kinase C in intact cells was studied by treating cultures with phospholipase C. This treatment caused a decrease in phorbol ester binding consistent with the diacylglyerol generated being a competitive inhibitor of phorbol ester binding. Phospholipase C-treatment mimicked phorbol ester effects as measured by redistribution of receptors from the cytosol to the membrane compartments, decreased binding of epidermal growth factor, and increased secretion of prolactin.